Mass spectrometry (MS) is a sensitive procedure to measure the masses of particles and chemical compounds, such as biomolecules like proteins, peptides, DNA. However, biomolecules require specialized techniques to enable desorption and ionization of the molecules while keeping them intact, such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).
In the MALDI process, a matrix is used to protect and assist in ionization by donating charge to the analyte biomolecules when excited by the laser. The matrix consists of crystallized molecules specific to the type of analyte to be investigated. The most common matrices used today are based on benzoic or cinnamic acids and absorb light with wavelengths below 350 nm. In most sample preparation procedures, the matrix is first dissolved in the appropriate solvent (de-ionized water, various organic solvents, etc.), then mixed with solution containing the analyte molecules.
There are five main types of matrix, seen in FIGS. 1(A) through (E), with their use is determined by the type of molecule to be investigated. For investigating proteins, sinapinic (Beavis, & Bridson, Epitaxial Protein Inclusion in Sinapic Acid Crystals. Journal of Physics D-Applied Physics, 1993. 26(3): p. 442-447) and ferulic acids are typically used. For peptides, sinapinic acid, α-cyanohydroxycinnamic acid (CHCA), (Beavis, et al., Alpha-Cyano-4-Hydroxycinnamic Acid as a Matrix for Matrix-Assisted Laser Desorption Mass-Spectrometry. Organic Mass Spectrometry, 1992. 27(2): p. 156-158) and dihydroxy benzoic acid (DHB) (Strupat, et al., 2,5-Dihydroxybenzoic Acid—a New Matrix for Laser Desorption Ionization Mass-Spectrometry. International Journal of Mass Spectrometry and Ion Processes, 1991. 111: p. 89-102) are used. For oligonucleotides and DNA probes, DHB and hydroxy picolinic acid (HPA) are used. The natural state of the matrix molecules is a crystalline form, and is dissolved in an appropriate solvent (typically highly purified water and an organic solvent such as acetonitrile) for use.
There are two main types of sample preparation, sequential deposition in which the matrix solution is deposited on the MALDI plate and allowed to crystallize before the analyte solution is deposited on top (Dai, et al., Two-layer sample preparation: A method for MALDI-MS analysis of complex peptide and protein mixtures. Analytical Chemistry, 1999. 71(5): p. 1087-1091), and concurrent deposition where the matrix and analyte solutions are mixed before deposition. In the latter technique, the analyte is distributed throughout the matrix and is said to be co-crystallized. The sequential deposition technique has the advantage of increased analyte concentration on the surface of the matrix crystals, if the matrix is not entirely re-dissolved upon analyte application. However, sample homogeneity is affected due to the lack of analyte in the bulk of the matrix crystals, leading to signal degradation over time with increased laser investigation of the same sample spot. The co-crystallization technique produces matrix crystals with a more uniform concentration of embedded analyte, which produces more consistent ion signals over time.
The resultant solution is deposited onto a MALDI plate, where the matrix re-crystallizes with the analyte as the solvents evaporate thereby forming a spot, through a process called co-crystallization.
For analysis, the plate is loaded into the MALDI instrument and subjected to a vacuum, followed by laser stimulation. It should be noted that atmospheric pressure MALDI is also possible, but has limitations in sensitivity and mass range. The energy from the laser is absorbed by the matrix, which transfers charge to the analyte, generating plumes of both matrix and analyte molecules that are desorbed from the plate surface. Ideally, the matrix should desorb from the sample surface without destructively heating the analyte. Ionization is assumed to occur at the sample surface and in the initial stages of the resulting plume of molecules. The ionized analyte molecules are detected by a time-of-flight (TOF) mass spectrometer and the data is plotted in a graph of intensity vs. mass-to-charge ratio.
When the matrix molecules reach the desorption temperature, which is based on the matrix, the molecules are liberated from the sample surface at velocities above 600 m per second. Because the desorption is primarily dependent on the electron excitation, increasing the laser fluence considerably above the plume generation threshold can lead to excessive matrix desorption and increased noise in a sample spectra. The energy and diameter of the laser used for plume generation can be factors in instrument resolution as a result of differing plume dynamics. Additionally, the expansion of the matrix plume might be influenced by charges that the molecules carry as a result of being excited by the primary laser.
Evaporation speed is also a factor in sample preparation. It has been observed that fast evaporation produces high density fields of smaller crystals (Beeson, et al., Aerosol Matrix-Assisted Laser-Desorption Ionization-Effects of Analyte Concentration and Matrix-to-Analyte Ratio. Analytical Chemistry, 1995. 67(13): p. 1981-1986). This can lead to an increase in surface area available for laser absorption and analyte desorption compared to the larger matrix crystals that are obtained through slow evaporation. Crystal density on the sample spot can have an effect on the MALDI spectra. An increased number of crystals in contact can increase the energy pooling efficiency resulting in increased analyte ionization at the crystal surface.
However, in current sample preparation the dried mixture of matrix and analyte has been shown to be non-homogenous, most likely a result of separation of the two substances during crystallization. This increases the number of investigations required to get an accurate average of the analyte signal.
Droplet deposition on traditional metal substrates resulted in nonhomogeneous dried droplets with a majority of the matrix crystals forming a ring around the edge and a central area that was either blank or contained microcrystals, which is consistent with the results found in this work when using this technique. One of the techniques designed to decrease sample spot size is the use of patterned areas using hydrophobic and hydrophilic materials. Schuerenberg (Schuerenberg, et al., Prestructured MALDI-MS sample supports. Analytical Chemistry, 2000. 72(15): p. 3436-3442) published results using a substrate composed of a hydrophobic Teflon® field with an array of hydrophilic gold spots to act as sample anchors. The sample supports used in the experiment consisted of a stainless steel sample plate coated with polytetrafluoroethylene (PTFE, or commonly known as Teflon®) to a thickness between 30-40 μm. Gold spots were deposited via sputtering to a thickness of 30 nm, with spot diameters ranging from 100-300 μm (Schuerenberg, et al., Prestructured MALDI-MS sample supports. Analytical Chemistry, 2000. 72(15): p. 3436-3442), which are considerably smaller than the diameter of the samples prepared using the traditional dried droplet technique (approaching 1 mm diameter, depending on solution composition, concentrations, and deposition volume). Since the typical laser used in MALDI-MS instruments has a diameter typically between 100-200 μm, the objective of the technique is lateral concentration of the sample onto the anchor spot. Solutions containing matrices 2,5-dihydroxybenzoic acid (DHB) or 3-hydroxypicolinic acid (3-HPA) were deposited via pipette onto the patterned 200 μm. When 3-HPA matrix-samples exceeded the gold spot diameter, thought to be a result of excess matrix material in solution. The author also reported in several instances the shrinking sample droplet would leave the gold spot and crystallize on the surrounding Teflon surface. Investigations with the 300 μm diameter gold spots revealed that the drying crystals resulted in structures that resembled those on plain metal, with a thicker crystalline rim and a blank center area. This was thought to be a result of insufficient matrix concentration in solution, as this phenomenon was avoided by increasing the matrix concentration previous to droplet deposition (Schuerenberg, et al., Prestructured MALDI-MS sample supports. Analytical Chemistry, 2000. 72(15): p. 3436-3442). As such, matrix composition and concentration are critical for successful concentration during drying.
Carbon nanotubes (CNTs) are one of the many different occurring forms (or allotropes) of the element carbon, comprising tubular structures composed entirely of carbon atoms that are joined with 120° bond angles that resemble rolled up sheets of grapheme (Dresselhaus, et al., Carbon-Fibers Based on C-60 and Their Symmetry. Physical Review B, 1992. 45(11): p. 6234-6242). CNT's can be single tube (single-walled) or multiple tubes inside each other (multi-walled), according to the type of growth process. Each wall can be classified as “armchair”, “zigzag” or “chiral” depending on the orientation of the carbon bond angles with respect to the diameter of the tube (Dresselhaus, et al., Physics of Carbon Nanotubes. Carbon, 1995. 33(7): p. 883-891). Each type has characteristic properties such as minimum diameter and electron conductivity. The conductivity of the carbon nanotube is also determined by the chiral vector (Saito, R., et al., Electronic-Structure of Chiral Graphene Tubules. Applied Physics Letters, 1992. 60(18): p. 2204-2206). Depending on the lattice unit cell, CNTs can behave as a metal or semiconductor. CNTs with armchair structure behave as a metal, while zigzag and chiral CNTs can behave as either a metal or semiconductor depending on the vector. For carbon nanotubes that consist of multiple walls, each wall can have its own chiral vector and electronic properties. However in practice, multi-walled CNTs usually display metallic properties, as one of the shells has a chiral vector consistent with metallic properties, hence dominating conduction when the entire nanotube is measured.
Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) is a technique used for the quantification and detection of bio-molecules and other macro-molecular substances for applications ranging from proteomics and cancer early detection to forensic investigations. Especially for proteomics and cancer research, sensitivity and analyte concentration are essential for successful measurements, since the analyte is often only available in very small quantities and/or high dilution.
The described invention aims at increasing the reproducibility and sensitivity of MALDI-MS for water-insoluble matrix based samples through improving the sample preparation process. MALDI-MS is an advanced mass spectrometry technique used to detect large molecules (“macro-molecules”). Such molecules cannot be measured using conventional mass spectrometry techniques due to fragmentation. MALDI-MS achieves ionization by proton transfer from a matrix compound (usually a crystal-forming acid) to the analyte to be analyzed. To achieve this proton transfer, the analyte needs to be embedded within the matrix compound, which generally exceeds the analyte amount by two to three magnitudes. This is achieved by creating a mixed solution of both analyte and matrix, which is drop-deposited on a sample plate. Evaporation results in a solid residue of analyte/matrix compound. This solid residue is then ablated with a laser focused into a tight (˜100 μm diameter) high-intensity spot. The ablated material forms a gaseous cloud above the sample in which protons are transferred from matrix to analyte, resulting in charging of the analyte molecules, which can subsequently be analyzed in the mass spectrometer by use of electrical or magnetic fields.
The standard matrix materials used in MALDI investigations can be generally divided into water-soluble and water-insoluble compounds. The mostly used water-soluble compounds are 2,5-dihydroxybenzoic acid (2,5-DBH) and 3-hydroxypicolinic acid (3-HPA), while the most popular water-insoluble material is α-cyano-4-hydroxycinnamic acid (HCCA).
Since drop-deposition of matrix/analyte solution on a flat plate typically yields irregular circular deposits, such deposits are difficult to analyze. Usually a trial and error procedure is used to find a “sweet spot” that yields a good signal-to-noise ratio. This is time consuming, and yields poorly reproducible data. Consequently, this has led to the invention of so-called anchor plates, where arrays of small (100-800 μm diameter) hydrophilic spots are created on a hydrophobic substrate. This allows deposited droplets to anchor to the hydrophilic spots, since they are repelled by the hydrophobic surroundings. Successively, evaporation results in crystallization of the matrix/analyte deposit on or close to the hydrophilic spot. This allows a much more reproducible interrogation of the sample since the laser spot covers a larger portion of the area coated with the matrix/analyte deposit. This eliminates the hunt for the “sweet spot”, while also increasing the sensitivity of the measurement due to the analyte concentration effect of the procedure (Schuerenberg, C. Luebbert, H. Eickhoff, M. Kalkum, H. Lehrach and E. Nordhoff: “Prestructured MALDI-MS sample supports”, Analytical Chemistry 72 (15), pp. 3436-3442 (2000).).
This procedure works reliably with water-soluble matrix compounds such as 2,5-DHP or 3-HPA, while it does not work well with water-insoluble matrix compounds such as HCCA (Schuerenberg, C. Luebbert, H. Eickhoff, M. Kalkum, H. Lehrach and E. Nordhoff: “Prestructured MALDI-MS sample supports”, Analytical Chemistry 72 (15), pp. 3436-3442 (2000); M. Schuerenberg: “AnchorChip™ Technology, Revision 2.3”, Bruker Product Information, (2005)). When HCCA is used, the final deposit is spread over an area much wider than the anchor spot. The reason for this behavior lies in the necessity to use an organic solvent mixable with water to dissolve the HCCA matrix. Usually, acetonitrile is used as organic solvent since it dissolves HCCA, and it is polar enough to mix well with the aqueous solution containing the analyte to be investigated.
However, the current laser-based mass spectrometry supports are unable to reliably deposit or concentrate samples having micro-liter volume and suffer from high signal to noise ratios below a threshold analyte concentration. The present invention addresses these issues through a novel device designed to nucleate analyte at a specific, pre-determined location.